This invention relates to agarose compositions having improved properties, particularly low electroendosmosis (EEO), for use in electrophoresis and other diffusive procedures or interactions. The invention further relates to processes for purifying agaroses to improve their electrophoretic properties, and to methods of using the agaroses in electrophoresis and other applications.
The rapid expansion of interest in the purification and separation of biomolecules such as proteins and nucleic acids, the mapping of genes, and DNA sequencing, has placed increased demands on agaroses as separation media. Agaroses have been prepared commercially by the polyethylene glycol method of Polson (U.S. Pat. No. 3,335,127), the aluminum hydroxide adsorption method of Barteling, Clin. Chem. 15, 1002-1005 (1969), and the quaternary ammonium salt/sulfated polysaccharide method of Blethen (U.S. Pat. No. 3,281,409). In all of these methods the larger, least-charged agarose molecules are separated from the more highly charged agaropectin molecules.
Although agaroses prepared by these methods remain satisfactory for many electrophoretic, immunodiffusion and chromatographic applications, agaroses are now required which not only have high gel strength (to allow use at low concentrations - the larger pores permit separation of larger molecules), but also allow faster, more reliable and precise separations, and detection of minute quantities of material.
Various purification techniques have been developed in efforts to produce improved agaroses, beginning with the ion exchange work of Zabin (U.S. Pat. No. 3,423,396) and Duckworth and Yaphe (U.S. Pat. No. 3,753,972), and continuing with the work of Laas and co-workers (J. Chromatogr 60 (1971), 167-177 and 66 (1972), 3476-355; Anal. Biochem. 72 (1976), 527-532) on alkaline desulphation followed by alcohol precipitation and reduction with lithium aluminum hydride. However, these methods result in material which does not have reliable quality or require expensive and/or unsafe procedures. Exhaustive treatment with alkali will remove much of the sulfate but can also degrade the gel strength of agar and agarose.
For preparation of agaroses for use where low, essentially zero, EEO is important, as in isoelectric focusing, the residual EEO can be suppressed by addition of a gum such as clarified locust bean or guar gum (U.S. Pat. No. 4,290,911 to Cook and Witt) or charge balanced by introduction into the agarose of positively-charged groups (U.S. Pat. No. 4,312,739 to Hansson and Kagedal). When used in electrophoresis, however, these media tend to bind (immobilize) biomolecules such as DNA because of the added materials or groups. The media also have lower gel strength and the modifications adversely affect their gelling and melting temperatures.
Lai, Birren and colleagues, in studying various forms of pulsed field gel electrophoresis, determined that DNA moves fastest in gels prepared from agaroses of low EEO, thus relating speed of separation to EEO (BioTechniques 7, No. 1 (1989), 34-42, at 39). This relationship thus has become a partial measure of usefulness of agaroses for modern electrophoretic processes.